Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.


GM-CSF disruption in CART cells modulates T cell activation and enhances CART cell anti-tumor activity


Inhibitory myeloid cells and their cytokines play critical roles in limiting chimeric antigen receptor T (CART) cell therapy by contributing to the development of toxicities and resistance following infusion. We have previously shown that neutralization of granulocyte-macrophage colony-stimulating factor (GM-CSF) prevents these toxicities and enhances CART cell functions by inhibiting myeloid cell activation. In this report, we study the direct impact of GM-CSF disruption during the production of CD19-directed CART cells on their effector functions, independent of GM-CSF modulation of myeloid cells. In this study, we show that antigen-specific activation of GM-CSFKO CART19 cells consistently displayed reduced early activation, enhanced proliferation, and improved anti-tumor activity in a xenograft model for relapsed B cell malignancies. Activated CART19 cells significantly upregulate GM-CSF receptors. However, the interaction between GM-CSF and its upregulated receptors on CART cells was not the predominant mechanism of this activation phenotype. GM-CSFKO CART19 cell had reduced BH3 interacting-domain death agonist (Bid), suggesting an interaction between GM-CSF and intrinsic apoptosis pathways. In conclusion, our study demonstrates that CRISPR/Cas9-mediated GM-CSF knockout in CART cells directly ameliorates CART cell early activation and enhances anti-tumor activity in preclinical models.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Prices vary by article type



Prices may be subject to local taxes which are calculated during checkout

Fig. 1: GM-CSF directly contributes to CART cell activation and apoptosis.
Fig. 2: GM-CSF knockout via CRISPR/Cas9 does not impair CART19 production and effector functions.
Fig. 3: GM-CSF disruption on CART19 modulates early activation and anti-tumor activity.
Fig. 4: Activated T cells and CART19 cells express high levels of GM-CSF receptors.
Fig. 5: GM-CSFKO CART19 are intrinsically more resistant to apoptosis.

Data availability

Sequencing data are available at BioProject PRJNA623000.


  1. June CH, Sadelain M. Chimeric antigen receptor therapy. N Engl J Med. 2018;379:64–73.

    Article  CAS  Google Scholar 

  2. Anagnostou T, Riaz IB, Hashmi SK, Murad MH, Kenderian SS. Anti-CD19 chimeric antigen receptor T-cell therapy in acute lymphocytic leukaemia: a systematic review and meta-analysis. Lancet Haematol. 2020;7:e816–e826.

    Article  Google Scholar 

  3. Khadka RH, Sakemura R, Kenderian SS, Johnson AJ. Management of cytokine release syndrome: an update on emerging antigen-specific T cell engaging immunotherapies. Immunotherapy. 2019;11:851–7.

    Article  CAS  Google Scholar 

  4. Teachey DT, Lacey SF, Shaw PA, Melenhorst JJ, Maude SL, Frey N, et al. Identification of predictive biomarkers for cytokine release syndrome after chimeric antigen receptor T-cell therapy for acute lymphoblastic leukemia. Cancer Discov. 2016;6:664–79.

    Article  CAS  Google Scholar 

  5. Locke FL, Ghobadi A, Jacobson CA, Miklos DB, Lekakis LJ, Oluwole OO, et al. Long-term safety and activity of axicabtagene ciloleucel in refractory large B-cell lymphoma (ZUMA-1): a single-arm, multicentre, phase 1–2 trial. Lancet Oncol. 2019;20:31–42.

    Article  CAS  Google Scholar 

  6. Schuster SJ, Svoboda J, Chong EA, Nasta SD, Mato AR, Anak O, et al. Chimeric antigen receptor T cells in refractory B-cell lymphomas. N Engl J Med. 2017;377:2545–54.

    Article  CAS  Google Scholar 

  7. Sakemura R, Cox MJ, Hefazi M, Siegler EL, Kenderian SS. Resistance to CART cell therapy: lessons learned from the treatment of hematological malignancies. Leuk Lymphoma. 2021;62:1–18.

    Article  Google Scholar 

  8. Cox MJ, Kenderian SS. Omics analyses provide insights to CART cell therapy resistance. J Transl Genet Genom 2021;5:80–8.

    CAS  Google Scholar 

  9. Sterner RM, Kenderian SS. Myeloid cell and cytokine interactions with chimeric antigen receptor-T-cell therapy: implication for future therapies. Curr Opin Hematol. 2020;27:41–48.

    Article  CAS  Google Scholar 

  10. Stroncek DF, Ren J, Lee DW, Tran M, Frodigh SE, Sabatino M, et al. Myeloid cells in peripheral blood mononuclear cell concentrates inhibit the expansion of chimeric antigen receptor T cells. Cytotherapy. 2016;18:893–901.

    Article  CAS  Google Scholar 

  11. Ruella M, Klichinsky M, Kenderian SS, Shestova O, Ziober A, Kraft DO, et al. Overcoming the immunosuppressive tumor microenvironment of Hodgkin Lymphoma using chimeric antigen receptor T cells. Cancer Disco. 2017;7:1154–67.

    Article  CAS  Google Scholar 

  12. Jain MD, Zhao H, Wang X, Atkins R, Menges M, Reid K, et al. Tumor interferon signaling and suppressive myeloid cells associate with CAR T cell failure in large B cell lymphoma. Blood. 2021;137:2621–33.

    Article  CAS  Google Scholar 

  13. Neelapu SS, Locke FL, Bartlett NL, Lekakis LJ, Miklos DB, Jacobson CA, et al. Axicabtagene ciloleucel CAR T-cell therapy in refractory large B-cell lymphoma. N Engl J Med. 2017;377:2531–44.

    Article  CAS  Google Scholar 

  14. Shi Y, Liu CH, Roberts AI, Das J, Xu G, Ren G, et al. Granulocyte-macrophage colony-stimulating factor (GM-CSF) and T-cell responses: what we do and don’t know. Cell Res. 2006;16:126–33.

    Article  CAS  Google Scholar 

  15. Tugues S, Amorim A, Spath S, Martin-Blondel G, Schreiner B, De Feo D, et al. Graft-versus-host disease, but not graft-versus-leukemia immunity, is mediated by GM-CSF-licensed myeloid cells. Sci Transl Med. 2018;10.

  16. Sterner RM, Sakemura R, Cox MJ, Yang N, Khadka RH, Forsman CL, et al. GM-CSF inhibition reduces cytokine release syndrome and neuroinflammation but enhances CAR-T cell function in xenografts. Blood. 2019;133:697–709.

    Article  CAS  Google Scholar 

  17. Sterner RM, Cox MJ, Sakemura R, Kenderian SS. Using CRISPR/Cas9 to knock out GM-CSF in CAR-T cells. J. Vis. Exp. 2019;149:e59629.

    Google Scholar 

  18. Manriquez-Roman C, Siegler EL, Kenderian SS. CRISPR takes the front seat in CART-cell development. BioDrugs. 2021;35:113–24.

    Article  Google Scholar 

  19. Sachdeva M, Duchateau P, Depil S, Poirot L, Valton J. Granulocyte-macrophage colony-stimulating factor inactivation in CAR T-cells prevents monocyte-dependent release of key cytokine release syndrome mediators. J Biol Chem. 2019;294:5430–7.

    Article  CAS  Google Scholar 

  20. Zhang J, Roberts AI, Liu C, Ren G, Xu G, Zhang L, et al. A novel subset of helper T cells promotes immune responses by secreting GM-CSF. Cell Death Differ. 2013;20:1731–41.

    Article  CAS  Google Scholar 

  21. Li H, Durbin R. Fast and accurate long-read alignment with Burrows–Wheeler transform. Bioinformatics. 2010;26:589–95.

    Article  Google Scholar 

  22. McKenna A, Hanna M, Banks E, Sivachenko A, Cibulskis K, Kernytsky A, et al. The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res. 2010;20:1297–303.

    Article  CAS  Google Scholar 

  23. Genomes Project C, Auton A, Brooks LD, Durbin RM, Garrison EP, Kang HM, et al. A global reference for human genetic variation. Nature. 2015;526:68–74.

    Article  Google Scholar 

  24. Stemmer M, Thumberger T, Del Sol Keyer M, Wittbrodt J, Mateo JL. CCTop: an intuitive, flexible and reliable CRISPR/Cas9 target prediction tool. PLoS One. 2015;10:e0124633.

    Article  Google Scholar 

  25. Brinkman EK, Chen T, Amendola M, van Steensel B. Easy quantitative assessment of genome editing by sequence trace decomposition. Nucleic Acids Res. 2014;42:e168–e168.

    Article  Google Scholar 

  26. Iyer V, Boroviak K, Thomas M, Doe B, Riva L, Ryder E, et al. No unexpected CRISPR-Cas9 off-target activity revealed by trio sequencing of gene-edited mice. PLoS Genet. 2018;14:e1007503.

    Article  Google Scholar 

  27. Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014;15:550.

    Article  Google Scholar 

  28. Zhang Y, Li H, Wang X, Gao X, Liu X. Regulation of T cell development and activation by creatine kinase B. PLoS One. 2009;4:e5000.

    Article  Google Scholar 

  29. Juric V, Chen CC, Lau LF. Fas-mediated apoptosis is regulated by the extracellular matrix protein CCN1 (CYR61) in vitro and in vivo. Mol Cell Biol. 2009;29:3266–79.

    Article  CAS  Google Scholar 

  30. Yamamoto TN, Lee PH, Vodnala SK, Gurusamy D, Kishton RJ, Yu Z, et al. T cells genetically engineered to overcome death signaling enhance adoptive cancer immunotherapy. J Clin Investig. 2019;129:1551–65.

    Article  Google Scholar 

  31. Michelozzi IM, Gomez-Castaneda E, Pohle RVC, Rodriguez F, Sufi J, Puigdevall P, et al. The Enhanced Functionality of Low-Affinity CD19 CAR T Cells Is Associated with Activation Priming and Polyfunctional Cytokine Phenotype. Blood 2020;136:52–53.

    Article  Google Scholar 

  32. Tschumi BO, Dumauthioz N, Marti B, Zhang L, Schneider P, Mach JP, et al. CART cells are prone to Fas- and DR5-mediated cell death. J Immunother Cancer. 2018;6:71.

    Article  Google Scholar 

  33. Benmebarek MR, Karches CH, Cadilha BL, Lesch S, Endres S, Kobold S. Killing mechanisms of chimeric antigen receptor (CAR) T cells. Int J Mol Sci. 2019;20:1283.

    Article  CAS  Google Scholar 

  34. Gomes-Silva D, Mukherjee M, Srinivasan M, Krenciute G, Dakhova O, Zheng Y, et al. Tonic 4-1BB costimulation in chimeric antigen receptors impedes T cell survival and is vector-dependent. Cell Rep. 2017;21:17–26.

    Article  CAS  Google Scholar 

  35. Kunkele A, Johnson AJ, Rolczynski LS, Chang CA, Hoglund V, Kelly-Spratt KS, et al. Functional tuning of CARs reveals signaling threshold above which CD8+ CTL antitumor potency is attenuated due to cell Fas-FasL-dependent AICD. Cancer Immunol Res. 2015;3:368–79.

    Article  Google Scholar 

  36. Siegler EL, Simone BW, Sakemura R, Tapper EE, Horvei P, Cox MJ, et al. Efficient gene editing of CART cells with CRISPR-Cas12a for enhanced antitumor efficacy. Blood. 2020;136:6–7.

    Article  Google Scholar 

  37. Ghezraoui H, Piganeau M, Renouf B, Renaud JB, Sallmyr A, Ruis B, et al. Chromosomal translocations are mediated by canonical NHEJ in human cells. Cancer Discov. 2014;4:OF12.

    Article  Google Scholar 

  38. Stadtmauer EA, Cohen AD, Weber K, Lacey SF, Gonzalez VE, Melenhorst JJ, et al. First-in-human assessment of feasibility and safety of multiplexed genetic engineering of autologous T cells expressing NY-ESO -1 TCR and CRISPR/Cas9 gene edited to eliminate endogenous TCR and PD-1 (NYCE T cells) in advanced multiple myeloma (MM) and sarcoma. Blood. 2019;134:49.

    Article  Google Scholar 

  39. Yi Y, Chai X, Zheng L, Zhang Y, Shen J, Hu B, et al. CRISPR-edited CART with GM-CSF knockout and auto secretion of IL6 and IL1 blockers in patients with hematologic malignancy. Cell Disco. 2021;7:27.

    Article  CAS  Google Scholar 

Download references


Mayo Clinic Cancer Center (SK).


Exact Foundation (SSK), Humanigen (SSK), Mayo Clinic Center for Clinical and Translational Science grant UL1TR002377 (SSK), Mayo Clinic Center for Individualized Medicine (SSK), National Comprehensive Cancer Network (SSK), National Institutes of Health grants K12CA090628 (SSK) and R37CA266344-01 (SSK), Department of Defense grant CA201127 (SSK), and Predolin Foundation (RS and SSK).

Author information

Authors and Affiliations



SSK formulated the initial idea; MJC, CMR, RS, and SSK designed experiments; MJC, CMR, RS, EET, ELS, and SS performed experiments; MJC, CMR, RS, and SS analyzed data; MJC, RM, and NS analyzed sequencing data; SAP and NEK contributed reagents; SSK supervised the study. All authors edited and approved the final version of the manuscript.

Corresponding author

Correspondence to Saad S. Kenderian.

Ethics declarations

Competing interests

SSK is an inventor on patents in the field of CAR immunotherapy that are licensed to Novartis (through an agreement between Mayo Clinic, University of Pennsylvania, and Novartis). MJC, RS, RMS and SSK are inventors on patents in the field of CAR immunotherapy that are licensed to Humanigen (through Mayo Clinic). MH and SSK are inventors on patents in the field of CAR immunotherapy that are licensed to Mettaforge (through Mayo Clinic). SSK receives research funding from Kite, Gilead, Juno, BMS, Novartis, Humanigen, MorphoSys, Tolero, Sunesis/Viracta, Leahlabs, and Lentigen. NEK receives research funding from Acerta Pharma, BMS, Pharmacyclics, MEI Pharma, and Sunesis. NEK has participated in Advisory Board meetings of Cytomx Therapy, Janssen, Juno Therapeutics, Astra Zeneca, and Oncotracker; and on DSMC for Agios and Cytomx Therapeutics. SAP receives research funding from Pharmacyclics, MorphoSys, Janssen, AstraZeneca, TG Therapeutics, BMS, AbbVie, and Ascentage Pharma. SAP has participated in Advisory Board meetings of Pharmacyclics, AstraZeneca, Genentech, Gilead, GlaxoSmithKline, Verastem Oncology, and AbbVie (he was not personally compensated for his participation). CD, DC and OA are employees of Humanigen.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Rights and permissions

Reprints and Permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Cox, M.J., Manriquez Roman, C., Tapper, E.E. et al. GM-CSF disruption in CART cells modulates T cell activation and enhances CART cell anti-tumor activity. Leukemia 36, 1635–1645 (2022).

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:

This article is cited by


Quick links